Antimicrobial agents and methods of use

ABSTRACT

The present application relates to novel antimicrobial compositions and methods of using said antimicrobial compositions for inhibiting and treating microbial growth, microbial infections, inflammatory diseases, viral diseases, cardiovascular diseases, diabetes and/or conditions that may be regulated or associated with microbial infections, such as cancer.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of PCT International Application No.PCT/US11/22150 filed Jan. 21, 2011 which claims priority to U.S.Provisional Patent Application No. 61/412,375 filed on Nov. 10, 2010,entitled “Antimicrobial Agents and Methods of Use,” and to U.S.Provisional Patent Application No. 61/297,609 filed on Jan. 22, 2010,entitled “Antimicrobial and Immunomodulatory Agents and Methods of Use,”of which are hereby incorporated by reference in their entirety.

TECHNICAL FIELD

The present application relates to novel antimicrobial compositions andmethods of using said antimicrobial compositions for inhibiting andtreating microbial growth, microbial infections, inflammatory diseases,viral diseases, cardiovascular diseases, diabetes and/or conditions thatmay be regulated or associated with microbial infections, such ascancer.

BACKGROUND

The formation of biofilms on the surface of medical devices is a seriousand increasing problem for the medical community. Biofilms form on manytypes of surfaces, composed of a wide variety of materials, includingcatheters and ports, metal surfaces such as implanted prostheticdevices, live tissue such as deep wound trauma areas, and oral tissuessuch as teeth, gum tissue and bone. A number of types of organisms canoriginate biofilms including bacteria and fungi. Further, while somebiofilms can be occupied by a single species, more commonly biofilmsconsist of an entire community of a variety of organisms. In some cases,even viruses can participate in the pathology generated by the biofilmcommunity by way of bacteriophages. Both gram-negative and gram-positivebacterial organisms as well as fungi can produce biofilms.

While occupying a biofilm, many organisms, especially pathogens, exhibita changed profile of sensitivities or resistances to antibiotics. This,coupled with the physico-chemical protections provided by the biofilm,make treating patients with biofilm infections very difficult. Theproblem is increasingly difficult as more organisms become antibioticresistant, even when means can be found to deliver an effective dose ofan antibiotic to the biofilm occupants.

An additional problem arises when trying to design an antimicrobialtreatment to destroy a biofilm infection utilizing either small moleculeagents or antibiotic agents that are strongly effective againstplanktonic forms of biofilm organisms. The problem lies in the inabilityof the antimicrobial agents, such as antibiotics, to penetrate thebiofilm due in part to the biofilm acting to protect the embeddedmicroorganisms by preventing or reducing the antibiotic diffusion, thusonly reaching the target organisms in lowered concentration. One meansby which this form of barrier could operate is to react with theincoming antimicrobial agent at or near the surface, converting it intoa different and potentially less lethal form. Another mechanism isphysiology-based, positing that the biofilm-bound organisms areessentially undergoing modified metabolic process, relative to theplanktonic counterparts, the modification of which reduces theirsusceptibility to the antibiotic agent. Thus, the design of effectiveantimicrobial agents has presented many challenges.

During a microbial infection, various cellular stress responses are alsotriggered, leading to tissue inflammation and immune cell activation.These immune events, in turn, may promote the development of and/orsustain pathways that underlie downstream disorders such as cancer.However, molecular events linking these processes are not wellunderstood, hindering efforts to uncover effective immune modulatorsthat may be useful for the treatment of downstream immune-associatedconditions.

BRIEF SUMMARY

The present application relates to the discovery of a novel combinationof ingredients that collectively are effective as an antimicrobialagent. Accordingly, the present application describes novel compositionsand methods of using the antimicrobial agent for inhibiting and treatingmicrobial growth, microbial infections as well as inflammatory diseases,viral diseases, cardiovascular diseases, diabetes and conditions thatmay be regulated or associated with microbial infections, such as canceror pre-cancerous conditions.

In one embodiment, the present application provides an antimicrobialagent comprising (a) water; (b) a low molecular weight alcohol; (c) aperoxide or peroxide-generating agent; and (d) a chelating agent.

In some embodiments, the alcohol in the antimicrobial agent comprisesethanol. In some embodiments, the alcohol is present in theantimicrobial agent at a concentration of from about 1% to about 95% byvolume. In other embodiments, the alcohol is present from about 20% toabout 60% by volume. In alternative embodiments, the alcohol is presentat about 50% by volume.

In some embodiments, the chelating agent in the antimicrobial agentcomprises ethylenediamine tetraacetic acid (EDTA) and its acids andsalts thereof. In some embodiments, the EDTA is present at aconcentration of from about 5 mg/mL to about 50 mg/mL. In otherembodiments, the EDTA is present at a concentration of about 10 mg/mL.

In some embodiments, the peroxide or peroxide-generating agent in theantimicrobial agent comprises hydrogen peroxide (H₂O₂). In someembodiments, the H₂O₂ is present at a concentration of from about 0.05%to about 40% by volume. In other embodiments, the H₂O₂ is present at aconcentration of from about 0.05% to about 10% by volume. In alternativeembodiments, the H₂O₂ is present at a concentration of about 1.5% byvolume.

In some embodiments, the antimicrobial agent further comprises aviscosity-increasing agent. In some embodiments, theviscosity-increasing agent comprises hydroxypropyl methylcellulose(HPMC).

In some embodiments, the antimicrobial agent is useful in reducing orinhibiting microbial growth, microbial infections, inflammatorydiseases, viral diseases, cardiovascular diseases, diabetes orconditions resulting from or associated with microbial growth orinfection. In some embodiments, the antimicrobial agent is useful intreating microbial growth, microbial infections, inflammatory diseases,viral diseases, cardiovascular diseases, diabetes or conditionsresulting from or associated with microbial growth or infection.

In another embodiment, the present application provides a method ofinhibiting or reducing microbial growth, comprising administering to asubject a therapeutically effective amount of an antimicrobial agentcomprising: (a) water; (b) a low molecular weight alcohol; (c) aperoxide or peroxide-generating agent; and (d) a chelating agent. Inanother embodiment, the present application relates to a method oftreating microbial growth, comprising administering to a subject atherapeutically effective amount of an antimicrobial agent comprising:(a) water; (b) a low molecular weight alcohol; (c) a peroxide orperoxide-generating agent; and (d) a chelating agent.

In another embodiment, the present application relates to a method ofinhibiting or reducing a microbial infection, comprising administeringto a subject a therapeutically effective amount of an antimicrobialagent comprising: (a) water; (b) a low molecular weight alcohol; (c) aperoxide or peroxide-generating agent; and (d) a chelating agent. Inanother embodiment, the present application relates to a method oftreating a microbial infection, comprising administering to a subject atherapeutically effective amount of an antimicrobial agent comprising:(a) water; (b) a low molecular weight alcohol; (c) a peroxide orperoxide-generating agent; and (d) a chelating agent.

In some embodiments, the microbial growth or microbial infection is dueto a microorganism selected from the group consisting of a bacterium, afungus, a protozoa and a virus.

In some embodiments, the methods are for treating microbial growth ormicrobial infection associated with a medical device.

In another embodiment, the present application relates to a method ofinhibiting or reducing an inflammatory condition or disease comprisingadministering to a subject a therapeutically effective amount of anantimicrobial agent comprising: (a) water; (b) a low molecular weightalcohol; (c) a peroxide or peroxide-generating agent; and (d) achelating agent. In another embodiment, the present application relatesto a method of treating an inflammatory condition or disease comprisingadministering to a subject a therapeutically effective amount of anantimicrobial agent comprising: (a) water; (b) a low molecular weightalcohol; (c) a peroxide or peroxide-generating agent; and (d) achelating agent.

In another embodiment, the present application relates to a method ofinhibiting or reducing a viral condition or disease comprisingadministering to a subject a therapeutically effective amount of anantimicrobial agent comprising: (a) water; (b) a low molecular weightalcohol; (c) a peroxide or peroxide-generating agent; and (d) achelating agent. In another embodiment, the present application relatesto a method of treating a viral condition or disease comprisingadministering to a subject a therapeutically effective amount of anantimicrobial agent comprising: (a) water; (b) a low molecular weightalcohol; (c) a peroxide or peroxide-generating agent; and (d) achelating agent.

In some embodiments, the methods for treating inflammatory or viralconditions or diseases are associated with microbial growth or microbialinfection. In some embodiments, the microbial growth or microbialinfection is associated with a medical device.

In some embodiments, the methods for treating inflammatory conditionsare associated with cardiovascular diseases, diabetes and cancer orpre-cancerous conditions. In other embodiments, the methods for treatingviral conditions are associated with cardiovascular diseases, diabetesand cancer or pre-cancerous conditions.

In another embodiment, the present application relates to a method ofinhibiting or reducing a condition or disease resulting from orassociated with microbial growth or infection comprising administeringto a subject a therapeutically effective amount of an antimicrobialagent comprising: (a) water; (b) a low molecular weight alcohol; (c) aperoxide or peroxide-generating agent; and (d) a chelating agent. Inanother embodiment, the present application relates to a method oftreating a condition or disease resulting from or associated withmicrobial growth or infection comprising administering to a subject atherapeutically effective amount of an antimicrobial agent comprising:(a) water; (b) a low molecular weight alcohol; (c) a peroxide orperoxide-generating agent; and (d) a chelating agent.

In some embodiments, the condition or disease resulting from orassociated with microbial growth or infection is selected from the groupconsisting of cancer or pre-cancerous conditions, inflammatory diseaseand viral disease. In some embodiments, the condition is cancer orpre-cancerous conditions.

In another embodiment, the present application relates to a method ofinhibiting or reducing an immune response comprising administering to asubject a therapeutically effective amount of an antimicrobial agentcomprising: (a) water; (b) a low molecular weight alcohol; (c) aperoxide or peroxide-generating agent; and (d) a chelating agent.

In some embodiments, the methods of inhibiting or reducing an immuneresponse comprises administering the antimicrobial agent at an amounteffective in inhibiting local or systemic toxicity. In some embodiments,the antimicrobial agent is at an amount effective in inhibiting cytokineor chemokine levels or activity and/or cytokine or chemokine receptorlevels or activity. In some embodiments, the inhibition of cytokine orchemokine levels or activity is the result of chemical inhibition ormodification of the cytokine or chemokine and/or its receptor.

In some embodiments, the immune response is associated with cancer orpre-cancerous conditions, inflammatory disease, viral disease, microbialinfection, cardiovascular disease or diabetes. In some embodiments, theimmune response is associated with cancer or pre-cancerous conditions.In some embodiments, the microbial infection is due to a microorganismselected from the group consisting of a bacterium, a fungus, a protozoaand a virus.

In another embodiment, the present application relates to a method ofinhibiting or reducing biofilm formation, comprising: identifying asite; and applying an antimicrobial agent to the site, the antimicrobialagent comprising: (a) water; (b) a low molecular weight alcohol; (c) aperoxide or peroxide-generating agent; and (d) a chelating agent.

In some embodiments, the biofilm formation is the result of microbialgrowth or microbial infection. In some embodiments, the microbial growthor microbial infection is due to a microorganism selected from the groupconsisting of a bacterium, a fungus, a protozoa and a virus.

In some embodiments, the subject is human.

In some embodiments, the antimicrobial agent is administered by topicalapplication, intravenous injection, intraperitoneal injection orimplantation, intramuscular injection or implantation, intralesionalinjection (within a tumor), subcutaneous injection or implantation,intradermal injection, suppositories, pessaries, enteric application, ornasal route. In some embodiments, the agent is administered by topicalapplication.

In some embodiments, the antimicrobial agent is administered to a siteselected from the group consisting of a wound site, a catheter site, asurgical site, an injection site, a catheter, a catheter lumen, athermal burn site, a chemical burn site, a radiation burn site, a skinlesion, oral sites, bony sites, anal sites, vaginal sites, cervicalsites, vulvar sites, penile sites, ulcerated skin sites, acne sites,actinic keratosis sites, inflamed sites, irritated sites, gastric sites,gastrointestinal sites, esophageal sites, esophagogastrointestinalsites, intestinal sites, cardiac sites, vascular sites, nasal sites,nasopharyngeal sites, and aural sites.

This Brief Summary is provided to introduce simplified concepts relatedto antimicrobial compositions and methods of using said antimicrobialcompositions, which are further described below in the DetailedDescription. This summary is not intended to identify essential featuresof the claimed subject matter, nor should it be used to limit the scopeof the claims.

DETAILED DESCRIPTION OF THE APPLICATION

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this application belongs.

As used herein, the terms “subject,” “patient” and “individual” are usedinterchangeably herein, and mean a mammalian (e.g., human) subject to betreated and/or to obtain a biological sample from.

As used herein, the term “sample” is used herein in its broadest sense.For example, a sample including polynucleotides, peptides, antibodiesand the like may include a bodily fluid, a soluble fraction of a cellpreparation or media in which cells were grown, genomic DNA, RNA orcDNA, a cell, a tissue, skin, hair and the like. Examples of samplesinclude biopsy specimens, serum, blood, urine, plasma and saliva.

As used herein, the term “safe and effective amount” refers to thequantity of a component which is sufficient to yield a desiredtherapeutic response without undue adverse side effects (such astoxicity, irritation, or allergic response) commensurate with areasonable benefit/risk ratio when used as described herein.

As used herein, the term “therapeutically effective amount” means anamount of a composition as described herein effective to yield thedesired therapeutic response.

The specific safe and effective amount or therapeutically effectiveamount will vary with such factors as the particular condition beingtreated, the physical condition of the patient, the type of mammal oranimal being treated, the duration of the treatment, the nature ofconcurrent therapy (if any), and the specific formulations employed andthe structure of the compounds or its derivatives.

As used herein, the term “treatment” is defined as the application oradministration of a therapeutic agent to a patient, or application oradministration of the therapeutic agent to an isolated tissue or cellline from a patient, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease, or the predisposition toward disease.For example, “treatment” of a patient in whom no symptoms or clinicallyrelevant manifestations of a disease or disorder have been identified ispreventive or prophylactic therapy, whereas clinical, curative, orpalliative “treatment” of a patient in whom symptoms or clinicallyrelevant manifestations of a disease or disorder have been identifiedgenerally does not constitute preventive or prophylactic therapy.

Compositions and methods similar or equivalent to those described hereincan be used in the practice or testing of the present application.Suitable compositions and methods are described below.

There is a need for identifying improved antimicrobial agents withimproved activity (and in some cases with reduced toxicity), for optimaltherapeutic use, and for developing therapeutically effective clinicalregimens for these antimicrobial agents. Furthermore, there is a needfor formulations that are useful in a variety of related clinicalindications. The present application meets such needs, and furtherprovides other related advantages.

The present application is based on a novel combination of ingredientsintended to act as an antimicrobial agent in medical applications. Theantimicrobial agent of the present application comprises at least threeingredients, and is designed such that all ingredients in theantimicrobial agent are compatible with being placed in small quantitieswithin a human or animal patient's body with no long term undesirableeffects. The individual ingredients within the antimicrobial agent ofthe present application are known to be safe for application onto orinto the human or animal body in at least low levels. The presentapplication relates to a novel combination of the individual ingredientswhich provide a significant level of antimicrobial, antibacterial,antifungal, anti-inflammatory or antiviral action, or antibiofilm orimmunomodulatory action or some combination of these properties.

Biofilm Formation

The formation of biofilms on many types of surfaces is a serious andincreasingly unmet medical problem. It can form on many surfacesincluding catheters and ports, metal surfaces such as implantedprosthetic devices, live tissue such as deep wound trauma areas and oraltissues such as teeth, gum tissue and bone, to name but a few.

The development of antibiotic resistance by many of the microorganismsoccupying biofilms complicates the design of effective therapeutics. Inaddition, penetration into the biofilm by an agent poses a significanthurdle. Two scenarios have been postulated to attempt to explain themechanisms underlying the penetration into biofilms and the bacterialresistance that results. First is a transport-based explanation,suggesting that the biofilm is acting to protect the embeddedmicroorganisms by preventing or reducing the antibiotic diffusion, thusonly reaching the target organisms in reduced concentration. One meansby which this form of barrier could operate is to react with theincoming antimicrobial agent at or near the surface, converting it intoa different and potentially less lethal form. A second explanation isphysiology-based, positing that the biofilm-bound organisms areessentially undergoing modified metabolic process, relative to theplanktonic counterparts, the modification of which reduces theirsusceptibility to the antibiotic agents.

Quorum Sensing

Among the most significant advances in understanding of biofilms hasbeen the discovery of quorum sensing (QS) as the means by whichbiofilm-forming bacteria, communicate their own presence and initiatethe process of biofilm formation when their numbers reach a certainthreshold value. The search for the molecular-level communication“trigger” resulted in the identification of the family of acylhomoserine lactones (AHL's) as the primary quorum sensing communicationmolecule in gram-negative bacteria. In gram-positive bacteria, quorumsensing involves a cascade of at least three steps, but the predominantone, at least in some species is AHL. It is now understood that in bothgram-negative and gram-positive bacteria, detection of the QS signal isvia gene expression. When AHL is able to occupy the binding site of thesensing molecule, it begins the cascade of reactions which result in theproduction of the exopolysaccharide “slime” of the biofilm.

A similar family of molecules provides a quorum sensing function ingram-positive bacteria, although the studied cases are fewer. In atleast some cases, gram-positive bacteria use a three-step quorum sensingpathway, but the pathway involving AHL is still the primary feature. Inat least one case, Vibrio harveyi, a marine organism, the QS system iswell studied, and involves three parallel systems. Even in that morecomplex case, though the AHL system is primary. In that specific case,the AHL variant is N-(3-hydroxybutanoyl)homoserine lactone.

The AHL's all posses the lactone ring and the 3-position N-atom.Variations in the structure occur primarily in the N-bound chain,varying size, shape, chain length, saturation, and the presence orabsence of hetero-atoms. Variation between species occurs. Among thesevariations are degrees of saturations in the N-bound side chain. A fewcases are known wherein the side chain is cyclic. There is evidence thatindicate that AHL's bind to the active site of a trigger molecule whichin turn initiates the production of exopolysaccharide (EPS), the majorbuilding block of biofilms. Included in the evidence is that a series of3-substituted furanones have been shown to be strong antagonists of theAHL binding. These furanones are structurally similar to the AHL's, ofcourse, some occur naturally and some are synthetic, some arehetero-atom substituted for example with Br and are 3-substituted withside-chains of approximately similar structure. Often, within thefuranone ring structures are unsaturations (i.e., C═C bonds), which arenot directly similar to the lactone ring of AHL. Thesering-unsaturations do not prevent the AHL antagonism of the furanone.Because the furanone's key structural component is its ring rather thanits side chain, changes in side-chain do not cause large changes in theantagonism, while changes, such as for example halogen substitution inthe ring's substituents do.

A second indicative feature of the furanone behavior is that thefuranone is generally unable to completely block the AHL binding. Thisis generally considered to indicate a competitive binding between nativeAHL and the receptor, or between a similar structure of the (non-native)furanone and the receptor. If that be the case, the competitive bindingis most likely to also be reversible although to our knowledge that hasnot been established.

Assuming that the binding is reversible in both cases, these equilibria,shown in Eqn. 1 and Eqn. 2 would apply: We let [LuX-1 initiator]=1LuX 1 initiator+AHL

LuX 1−AHL  Eqn. 1:K _(AHL)=[LuX 1−AHL]/[AHL]Lux 1 inducer+Fur

LuX 1−Fur  Eqn. 2:K _(Fur)=[LuX 1−Fur]/[Fur] where Kfur˜1

The assignment of the K-value of the Kfur is approximate, based onvalues for similar reactions. The design of the antimicrobial agent ofthe present application took into consideration the desired reactionbetween alcohol (i.e., particularly ethanol (EtOH)) and AHL and whichwould be effective in the destruction of biofilms. The antimicrobialagent of the present application comprises an advantageous mixture ofEtOH, hydrogen peroxide (H₂O₂), and a chelator, specificallyethylenediamine tetraacetic acid (EDTA). Thus, the antimicrobial agentof the present application has at least two features known to beanti-biofilm, simply based on its composition.

EDTA has been reported to disrupt some biofilms, and EtOH is toxic to avariety of biofilm bacteria, including those inhabiting biofilms, aswell as those that are planktonic. In order to enhance the spectrum ofcases in which pathogenic organisms are destroyed, the antimicrobialagent of the present application will also inhibit or prevent biofilmformation. This strategy will improve the killing spectrum of ourantimicrobial agent by inhibiting biofilm formation, thus keeping agreater percentage of bacteria in the planktonic state, i.e., keepingthem in the state in which they are more vulnerable to killing byantibacterial or antibiotic agents. One means by which this goal mightbe achieved is to interrupt the QS system which, in turn, prevents thetransition from the planktonic to the biofilm state.

Our approach to AHL disruption is different from previous work. Ratherthan trying to develop an enzyme blocking substance that specificallyoccupies the AHL-binding site, we opted for a change in the chemicalstructure of AHL itself. If the content of AHL is never allowed to reachthe critical level required for QS, all the bacteria will stayunprotected and vegetative.

There are several well-known reactions which might allow thenon-oxidative disruption of the AHL molecule at mild temperatures,near-neutral pH and in an alcoholic aqueous solution such as theantimicrobial agent of the present application.

Option 1: Ring-opening hydrolysis at the lactone function:AHL+H₂O<=>HO—(CH₂)₂—CH—(CONH—R)—COOH

Option 2: Ring opening ethanolysis (i.e., transesterification) at thelactone function:AHL+EtOH<=>HO—(CH₂)₂—CH—(CONH—R)—COOEt

Option 3: Hydrolysis of the amide function in the side chain by water:AHL+H2O<=>3-amino tetrahydrofuran-2-one+HOOC—R (generally a fatty acid.)

All these reactions are equilibria, whose specific equilibrium constantsare not readily found. However, several chemical principles can be usedto estimate their reaction parameters to determine if they provide theneeded entry into biofilm prevention or inhibition. First, even if theequilibrium constant of the hydrolysis is unfavorably small, e.g., ifthe AHL structure is favored by the thermodynamics or kinetics of thering-opening hydrolysis or ethanolysis, reasonable amounts of reactionmight occur because of the large difference in concentrations. AHL's areknown to be active in nanomolar (nM) concentrations. On the other hand,a typical antimicrobial agent of the present application might contain,for example, 20-60% EtOH in water by volume. The density of thesesolutions is near 1.0-1.2 g/mL. This suggests that a 60% EtOH solutionwould contain about 500-600 g/L of EtOH, i.e., about 11 M in EtOH andabout 28-30 M in water. These levels exceed the AHL concentration byfactors of 10⁹. Thus, even if the equilibrium constant is veryunfavorable, small but likely sufficient amounts ofhydrolysis/ethanolysis would be expected at equilibrium.

However, the second phase reaction, namely oxidative consumption of thehydrolysis/ethanolysis product can occur, since H₂O₂ is present in theantimicrobial agent of the present invention at levels typically around3-6% (around 1.5 M-3.0 M). Peroxide reactions are often driven by fastkinetics, generally based on reactive free radicals, and/or by thephysical escape of a reaction product, for example by emission of a gas.Thus, in such a situation, the equilibrium between unmodified AHL andeither water or ethanol would be expected to shift towards thehydrolyzed or ethanolysed product as its equilibrium partner is rapidlyconverted to another species. AHL is thus removed from the solution orits concentration is so lowered as to hold it to levels below thecritical QS threshold. Therefore, QS will be interrupted, even with highinoculums of bacteria present.

Antimicrobial Agents of the Present Application

The antimicrobial agents of the present application comprise an alcohol,a peroxide or peroxide-generating agent and a chelating agent, with theremaining balance being made up of water. The unique design feature ofthe antimicrobial agent of the present application and the synergisticeffects derived from the combination of the individual componentsprovide a spectrum of effects that avoid the pitfalls ofsingle-component treatments.

The antimicrobial agents of the present application comprise an alcohol,preferably a low molecular weight alcohol. It can be present at aconcentration of from about 1% to about 95% by volume, preferably fromabout 20% to about 60% by volume, and more preferably at about 50% byvolume. Exemplary alcohols that are contemplated within the presentapplication include but are not limited to ethanol, isopropyl alcohol,n-propyl alcohol, butanol, pentanol, phenol and phenol derivatives,furanol and furanol derivatives, diols, triols, polyols, includingchain, ring and aromatics, and the like. An antimicrobial agentcomprising ethanol, and preferably about 50% by volume, is preferred.

The antimicrobial agents of the present application also comprise aperoxide or peroxide generating agent. It is present at a concentrationof from about 0.05% to about 40% by volume, preferably from about 0.05%to about 10% by volume, and more preferably at about 1.5% by volume.Exemplary peroxide or peroxide-generating agents that are contemplatedwithin the present application include but are not limited to hydrogenperoxide H₂O₂, carbamide peroxide (i.e., urea peroxide), peroxy acidssuch as peroxyacetic acid, peroxybenzoic acid, acetic anhydride, and thelike. In some cases, free-hydroxyl or free-radical generating substancescould be present or a substitute for hydrogen peroxide. Exemplaryfree-radical generating substances that are contemplated within thepresent application include but are not limited to acetone peroxide,t-butyl peroxide, di-t-butyl diazine ((t-Bu)₂N₂), and the like. Otherfree-radical generating materials include those which generate freeradicals on exposure to, for example, UV light. An antimicrobial agentcomprising H₂O₂, and preferably about 1.5% by volume is preferred.

The antimicrobial agents of the present application further comprise oneor more compounds that are chelating agents (chelators). They arepresent in the antimicrobial agents of the present application in theform which results when the pH of the solution is adjusted to thedesired level for a particular application. Exemplary chelating agentsthat are contemplated within the present application include but are notlimited to ethylenediamine tetraacetic acid (EDTA), citrate, and theirsalts, other substituted compounds, such as salicylic acid or salicylateesters, and the like. An antimicrobial agent comprising EDTA, andpreferably at a concentration of from about 5 mg/mL to about 50 mg/mL,more preferable at a concentration of about 10 mg/mL is preferred.

While not wishing to be limited by any particular theory, chelators areprimarily known to act to form strong bonds to a wide variety ofinorganic or organic ions, thereby rendering them relatively unavailablefor use in metabolic processes of various kinds. Specifically, said ionsare thereby prevented from binding by or use by certain proteins and/orenzyme systems to support or cause specific processes in metabolicaction. Exemplary proteins known to bind various ions are metalmetalloproteinases (MMPs), which bind divalent cations such as zinc(Zn+2). The chelating effect of agents such as EDTA may inhibit theactivity of MMPs by depriving the MMP of the Zn+2 ion (which is requiredfor its function). Thus, the chelating agents (such as EDTA) in theantimicrobial agents of the present application may assist incontrolling, inhibiting or avoiding tissue destruction caused by MMPs.Metallocarbamases also require divalent cations, e.g., zinc, and may beanother target for EDTA or similar chelators. Other metalloproteasesalso are contemplated.

Zinc finger proteins are generally found as DNA binding protectiveproteins. They contain one or more short loop(s) with a conservedHis-Cys motif binding generally one zinc ion per loop. They provide theDNA protective function by enwrapping the DNA molecule with a protein“glove” which it turn is held in place by one or more intercalated zincfingers spaced along the DNA helix. Generally, zinc finger formation andstability can be disrupted by chelators such as EDTA, by binding andcontrolling the amount of free Zn+2, thus providing a means by which theantimicrobial agents of the present application have the potential tomodify the degree of DNA protection from or exposure to other agents.

In addition, it is known that divalent ions, specifically Mg+2 and Ca+2ions, must be present for the formation and/or maintenance of thelipopolysaccharide matrix that forms the bulk of biofilms. It is furtherknown that EDTA imbues a solution with the power to disrupt orcompletely disintegrate an existing biofilm, and to retard or preventtheir formation at least in the case of some medically significantbiofilm forming organisms. It is therefore reasonable to suggest thatinclusion of EDTA or other chelators in the antimicrobial agent of thepresent application, especially along with the other active ingredients,will be synergistically useful in biofilm prevention and eradication.

In applications where the possibility exists that the antimicrobialagent of the present application might come in contact with a patient'sblood, it is desirable that the antimicrobial agent contain ananticoagulant. EDTA is often utilized in modern medical practice as ananticoagulant, for example as in blood draw tubes, e.g., Vacutainers®(Becton Dickinson and Company, Rutherford N.J.). Intravenous use is wellknown, e.g., in cases of metal chelation therapy to mitigate, amongother things, heavy metal (as for example, lead, Pb) poisoning. Theanticoagulant function of EDTA is thus an additional application to ourantimicrobial agent of the present application, especially inblood-contact situations. Another significant advantage of EDTAinclusion in the antimicrobial agent of the present application is thecase of a catheter lock solution, where contact with the patient'sbloodstream is assured. EDTA is compatible with IV therapy, being alsocommonly utilized in treatment for heavy metal poisoning.

EDTA can be used in various forms, for example, as the pure acid, or,for example, as the disodium salt, or for example, the calcium disodiumsalt, dipotassium salt, or tetrasodium salt. In all these cases andothers, the actual ionic composition of the EDTA in the antimicrobialagent of the present application will adjust, with the EDTA acting as abuffer, as the pH is adjusted. Furthermore, the EDTA provides aprotective function in the antimicrobial agent of the presentapplication solution, as in other known cases, by protecting theperoxide from divalent-catalyzed decomposition.

The present application provides the unexpected discovery that althoughEDTA has a relatively low solubility in solutions of, for example,ethanol, peroxide or peroxide-generating agents such as hydrogenperoxide (H₂O₂) can act as a powerful co-solvent. For example, insolutions of 50% v/v ethanol or higher, and at near-physiological pH butin the absence of H₂O₂, EDTA's solubility is limited to less than 10mg/mL. However, when H₂O₂ is present, even at levels as low as 1-2%,stable solutions of EDTA at 10 mg/mL and at least 50-60% v/v ethanol arereadily prepared and are stable (i.e., no precipitation or other changesare seen) at temperatures as low as 0° C. Furthermore, even in solutionsof 50% v/v ethanol, EDTA in the presence of 6% H₂O₂, remained soluble atconcentrations as high as 40 mg/mL, both at 27° C. and at 0° C. Thisunexpected effect is not due to the presence of additional waterintroduced with the H₂O₂, as evidenced by the fact that the effectoccurs even when the total amount of water in the solution is heldconstant.

Thus, the unique combination of the individual ingredients in theantimicrobial agent of the present application provides relatively highconcentrations of ethanol, high concentrations of EDTA and hydrogenperoxide levels that are unavailable by other means. The antimicrobialagent of the present application has unexpected stability, yet can befunctionally powerful and versatile as an antimicrobial agent. Stabilitystudies have shown shelf life lives (at room temperature) of 4% H₂O₂solutions with high ethanol concentrations (50%) and high EDTAconcentrations (10 mg/mL or greater) in excess of 14 months whileretaining essentially all of the peroxide and alcohol effectiveness. Theunexpected stability of EDTA against precipitation provided by the H₂O₂,and the stability of the hydrogen peroxide against decomposition,provided by EDTA is an example of synergy not normally seen in inanimatesystems and resembles a symbiosis relationship. Additional chelators canbe used to adapt the antimicrobial agent of the present application tospecific applications. Examples of these may include but are not limitedto dipicolinic acid, citrate, pyridine derivatives, various diamines orsubstituted diamines, and the like.

In addition to the unexpected stability, the relatively highconcentrations of alcohol (such as ethanol) in the antimicrobial agentof the present application is able to deliver its killing poweressentially unmitigated because of the unique properties conferred bythe peroxide agent (such as H₂O₂) and the chelating agent (such as EDTA)in the combination. For example, some evidence exists that suggestpenetration into a biofilm by H₂O₂ might be reduced because of thepresence of peroxide-reactive agents, e.g., catalase or peroxidase, nearthe outer (distal) surface of the biofilm and that only reducedconcentrations could penetrate deeply. The presence of ethanol in theantimicrobial agent of the present application can help mitigate thepotential reduction of H₂O₂ penetration by an effect discovered byMukergee et al. In some medical conditions, e.g., Candida albicans, thepresence of ethanol can significantly reduce the thickness of thebiofilm via the presence and action of the alcohol dehydrogenase (ADH)enzyme. The biofilm colonization of, for example, a catheter, isreduced. Further, ethanol is present in large concentration relative toboth the enzyme and its cofactor, NADH. The consumption of ethanol willbe severely limited, particularly because one of the mechanistic stepsin the alcohol dehydrogenase utilizes free Zn+2 ion. Thus, in thecombined action of the antimicrobial agent, EDTA—tying up zinc andsimultaneously attacking the biofilm structure itself with limited NADHcofactor for ADH—only a small fraction of the total ethanol will beconsumed, leaving the bulk remainder to act to penetrate the biofilmrapidly, delivering its killing power.

A viscosity-increasing agent (such as a thickener or gelling agent)might also be desirable. Exemplary viscosity-increasing agents includebut are not limited to carboxymethyl cellulose (CMC), hydroxypropylmethylcellulose (HPMC), methyl cellulose, methyl hydroxyethyl cellulose(MHEC), hydroxyethyl cellulose, sodium hydroxyalkyl celluloses, andadmixtures thereof. Other viscosity-increasing agents are contemplated,among them, but not limited to, silicone-based products such asdimethicone and silicone gels. An antimicrobial agent comprisinghydroxypropyl methylcellulose (HPMC), preferably at a concentration ofabout 0.7% by volume is preferred.

Additional ingredients may be included in the antimicrobial agent of thepresent application. It is usually necessary to adjust the pH of theantimicrobial agent for particular applications. For example, inapplications where the antimicrobial agent might be acidic onproduction, a base, typically but not exclusively sodium hydroxidesolution, can be added to adjust the pH to the desired pH or tophysiological pH. Alternatively, if the antimicrobial agent is basicwhen produced, an acid, typically but not exclusively eitherhydrochloric acid, citric acid, or acetic acid, can be added to returnthe pH to the desired pH or to physiological pH. In some embodiments, itmight be desirable for the antimicrobial agent to be at some non-neutralor non-physiological pH, in which case additional adjustments would bemade. In extreme cases, a far-from-neutral antimicrobial agent might beneeded, in which case an additional buffer might be needed. Such buffersare well known to practitioners of the art and a variety is availablefor use.

The balance of the antimicrobial agent will be made up of water.

The antimicrobial agent composition (such as strength of ingredients)can be tailored to the specific needs of an individual. For instance,the composition can be dependent upon such factors as nature of theinjury, depth of the wound, duration of time expired post injury,superinfection, vascularity and overall patient status (e.g., shock,renal failure, cardiorespiratory failure, coagulopathy). Alternatively,the antimicrobial agent of the present application can be applied inconjunction with medical dressings. Preferably, the dressing materialcan be a non-toxic material that will release the antimicrobial agentinto the medical areas as desired. Appropriate dressing materials willdepend upon the nature of the injury and the overall condition of thepatient.

The antimicrobial agent of the present application may be preferable insolution form in certain applications. In other applications, theantimicrobial agent of the present application may be preferable inother forms, such as gel, cream, ointment, drops, injection, spray,solid forms such as tablets, and the like.

The antimicrobial agent of the present application may be administeredas a bolus or as multiple doses over a period of time depending on theoverall condition of the patient and medical attention needed.

The antimicrobial agent of the present application may be administeredby many means including but not limited to topical application,intravenous injection, intraperitoneal injection or implantation,intramuscular injection or implantation, intralesional injection (e.g.,within a tumor), subcutaneous injection or implantation, intradermalinjection, suppositories, pessaries, enteric application, or nasalroute. Preferably, the antimicrobial agent of the present application isadministered by topical application.

The antimicrobial agent of the present application may be administeredto many areas including but not limited to a wound site (including skinaround wound areas), a catheter site, a surgical site, an injectionsite, a catheter, a catheter lumen, a thermal burn site, a chemical burnsite, a radiation burn site, a skin lesion (abrasions), oral sites (suchas leukoplakias, carcinomas-in-situ, oral carcinomas, “canker sores”(i.e., open lesions), bony sites (with osteomyelitis, for example,caused by Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacterbaumannii), anal sites, vaginal sites, cervical sites, vulvar sites,penile sites, ulcerated skin sites (e.g., diabetic foot ulcers, decubiti(“bed sore”) sites), acne sites (e.g., facial, trunkal, and others),actinic keratosis sites, inflamed sites, irritated sites, gastric sites,gastrointestinal sites (upper and lower), esophageal sites,esophagogastrointestinal sites, intestinal sites, cardiac sites,vascular sites, nasal sites, nasopharyngeal sites, and aural sites and acatheter locking solution.

Examples of Applications for the Antimicrobial Agents

The antimicrobial agent of the present application may be provided toinhibiting or reducing microbial growth. The antimicrobial agent of thepresent application may also be provided to inhibit or reduce microbialgrowth.

The antimicrobial agent of the present application may be provided toinhibiting or reducing microbial infections. The antimicrobial agent ofthe present application may also be provided to inhibit or reducemicrobial infections.

The microbial growth or microbial infections may be due to differentmicroorganism including but not limited to a bacterium, a fungus, aprotozoa and a virus. In certain applications, the microbial growth ormicrobial infection may be associated with a medical device includingbut not limited to catheters, stents, medical implants, dental devicesand implants, prosthetic devices and implants, and cardiac devices andimplants.

It is also contemplated that the antimicrobial agent of the presentapplication may be provided to certain critical surfaces external to thesubject or patient's body but in positions wherein ready access bypathogenic microorganisms to the subject or patient's tissues orbloodstream is available. Such sites include, without limitation,catheter lumens, catheter insertion sites, and catheter ports.Intravenous equipment and the like are also candidates for use. Inaddition, wounds, burns, skin lesions and vesicles, oral, cervical,vaginal, vulvar, penile, anal sites, esophageal sites, gastric sites,gastrointestinal sites, esophagogastrointestinal sites, intestinal sitesand the like are contemplated as intended use locations for theantimicrobial agent of the present application. For example, it iscontemplated that the antimicrobial agent of the present application maybe provided to such sites by endoscopy or radiological tube placementand intraluminal infusion.

The antimicrobial agent of the present application may be applied as animmunomodulator such that it interacts with matrix metalloproteinases(MMPs) or other cytokines or chemokines to modulate the degree ofinteraction between these naturally occurring substances and theunderlying tissues. Over expression of these naturally occurringsubstances may cause undesirable or harmful inflammation and otherdisturbances associated with healing or recovery from trauma or illness.

Accordingly, it is contemplated that the antimicrobial agent of thepresent application may be used to adjust or alter the amounts, levelsor activities of the MMPs, cytokine or chemokine, or TNF-alpha blockers,generally by a chemical interaction. For example, MMPs are so namedbecause they require the presence of at least one divalent metal ion,generally Zn+2, for their function. One component of the antimicrobialagent of the present application is designed to coordinate or chelatewith multivalent metal ions, thus preventing them from being availablefor use by the MMPs. For example, Tumor necrosis factor (TNF)-alphaconverting enzyme (TACE) is a MMP and the key sheddase that releasesTNF-alpha from its inactive cell-bound precursor. The activity of TACEmay be severely disrupted by the lack of multivalent metal ions thathave been sequestered by the chelating component of the antimicrobialagent of the present application. This, in turn, would reduce theamount, level and activity of active TNF-alpha and the associatedproinflammatory effects of TNF-alpha. Similarly, another component ofthe antimicrobial agent of the present application is designed toprovide an oxidative function to some portions of enzymatic molecules,providing a means by which they are inactivated or denatured. Thisoxidative function causes sulfur-bearing amino acids in the enzymestructures to be oxidized, a change which is reflected by changes in thecharge-bearing, the H-bonding, the solvent-bonding and hydrationproperties, and consequently, the overall folding configuration andshape of the protein, causing significant changes in its catalyticproperties. It is further contemplated that the oxidative component ofthe antimicrobial agent of the present application may causesulfur-bearing amino acids common in pro-inflammatory cytokines andchemokines (such as IL-8, IL-12, IL-6 and TNF-alpha) to be oxidized,which may significantly alter the levels and activities of theseproteins.

In addition to the synergistic effect on the chelation and oxidativefunctions provided by the antimicrobial agent components, it also hasthe ability to provide solvent interactions that can modify thehydration, the H-bonding, and the hydrophilic/hydrophobic balance of theoverall bio-environment. Such changes can be important in, for example,modifying various membranes that are important to pathogenic organisms,and perhaps even in modifying the structure of viruses or theirvirulence by modifying their target membranes of bacteria even ofeukaryotic cell targets, for example, the endoplasmic reticulum (ER) ofmitochondria. Similarly, such changes are also significant in that theycan effect, often by disrupting, the intercellular and/or theintracellular signaling of prokaryotes and eukaryotes, includingmulti-cellular organisms, including humans. In higher organisms,including humans, such chemical signaling can be involved in manyconditions including, for example, cancers, either in the carcinogenicstages, in the development of cancers or in the metastatic stages. Thearsenal of effects provided by the antimicrobial agent of the presentapplication is of value in controlling the chemical communication insuch organisms.

It is further contemplated that the antimicrobial agent of the presentapplication may adjust or alter the amounts, levels or activities of theMMPs, cytokines and chemokines via its nucleophilic interaction. Forexample, the EDTA in the antimicrobial agent of the present applicationmay play a role not only in the chelation of metal ions but may alsoexert nucleophilic activity attributable to its two amino groups. Thenucleophilic activity of the EDTA may result in disruption of peptidesof various biomolecules, particularly proteins such as cytokines andchemokine, and thereby disrupt their function. In addition, the S—Slinkage that creates the “loops” and “hairpin” turns in proteins mayalso be readily disrupted by nucleophiles, thereby altering the complex3-D structure and its actions. Proteins that contain the amino acidmethionine are also subject to 3-D structure modifications by theoxidative action of H₂O₂, readily converting the Met-S—R group to theMet-S(═O)—R group, which has significantly different polarity, shape andsolvent interactions. Thus, the antimicrobial agent of the presentapplication may modify and disrupt the structures of many biomoleculescausing significant changes to their activities and functions. Suchdisruption of, for example, MMP activity and function may significantlyaffect many downstream conditions, including, tumor formation, tumorprogression, tumor metastasis and angiogenesis. MMPs have been wellcharacterized as being key players in multiple steps of cancer, eitherat the carcinogenic stages, developmental stages or metastatic stages.

The antimicrobial agent of the present application may be used toinhibit or reduce an immune response, generally by inhibiting cytokineor chemokine levels or activity and/or cytokine or chemokine receptorlevels or activity. This can be achieved by many means including but notlimited to chemical inhibition or modification of the cytokine orchemokine and/or its receptor.

The antimicrobial agent of the present application may also provide ameans by which other potentially harmful components in a war wound,trauma, burn or other healing area are inactivated and tissue injury andsystemic toxicity are reduced. These components include but not limitedto host MMPs, TNF-alpha, bacterial beta-lactamases, (includingmulti-resistant extended spectrum beta-lactamases (ESBL)),carbapenemases, and metallocarbamases. The components are generallyrelatively complex protein-based compounds. However, the antimicrobialagents of the present application provides a spectrum of biochemicalreactions, at least one or more of which will be effective at disruptingand/or attenuating the harmful processes. Healing rates and/or thegeneral well-being of the subject will be improved by a reduction in theadverse effects seen from host over-expression of cytokines, chemokinesand other inflammatory molecules. Preservation of therapeutic activityof systemic antimicrobials, by preventing microbial enzymaticinactivation and lowering of bacterial and fungal toxins, will alsoaugment host survival.

Accordingly, the antimicrobial agent of the present application may alsobe provided to inhibit or reduce an inflammatory condition. Theantimicrobial agent of the present application may also be provided totreat an inflammatory condition. In some cases, the inflammatorycondition is associated with microbial growth or microbial infection. Inother cases, the inflammatory condition is associated with a medicaldevice.

The over-expression of cytokines, chemokines and other inflammatorymolecules during an inflammatory reaction induced as a result of, forexample, microbial infections, may further contribute to the initiation,development and/or progression of downstream conditions or diseases.

For example, chronic inflammation has also been associated with thedevelopment of cardiovascular diseases and related disorders. Studies ofthe inflammation paradigm in coronary pathogenesis suggest that chronicinfections may be involved by releasing cytokines and otherpro-inflammatory mediators (e.g., C-reactive protein (CRP), tumornecrosis factor (TNF-alpha)) that may in turn initiate a cascade ofbiochemical reactions and cause endothelial damage and facilitatecholesterol plaque attachment. Recent studies suggest that patients withelevated basal levels of C-reactive protein (CRP) are at an increasedrisk of cardiovascular disease (such as atherosclerosis), hypertensionand diabetes. CRP is an acute-phase protein found in the blood, thelevels of which rise in response to inflammation. It is commonly used asa marker of inflammation and infection.

There are also studies showing that periodontal diseases may increasethe risk of cardiovascular disease and that the risk is even greater forstroke. Epidemiological studies suggest that inflammation may be thelink between periodontal diseases and the cardiovascular complications.Interestingly, in patients with chronic periodontitis, elevated levelsof CRP have been detected in association with an increased risk ofdeveloping atherosclerosis. Periodontal therapy has been shown toproduce significant modulation of CRP levels and this may benefitindividuals with cardiovascular diseases. It is contemplated that theperiodontal therapy provided by the antimicrobial agent of the presentapplication would be effective in individuals with or at risk ofdeveloping cardiovascular diseases. Accordingly, the antimicrobial agentof the present application may be useful for treating cardiovasculardiseases and related disorders or reducing the risk of developingcardiovascular diseases and related disorders.

It has also been suggested that inflammatory activity may play a keypathogenic role in insulin resistance and diabetes. For example, theinflammatory biomarker CRP has been used to monitor insulin resistanceand cardiovascular risk in diabetic and nondiabetic individuals. Agrowing number of clinical trials have tested the hypothesis thatantidiabetic drugs specifically targeting insulin resistance couldbenefit individuals by reducing inflammation, atherogenesis, and thuscardiovascular risk. The clinical study results underline the benefit ofan early insulin resistance treatment to oppose systemic vascularinflammation and cardiometabolic syndrome in patients with elevatedlevels of CRP. Accordingly, the antimicrobial agent of the presentapplication may be useful for treating diabetes and related disorders orreducing the risk of developing diabetes and related disorders.

Chronic inflammation has also been shown to play an important role intumorigenesis, suggesting that negative regulation of inflammation islikely to be tumor suppressive. For example, one mediator that isinvolved in systemic inflammation and induces apoptotic cell death istumor necrosis factor (TNF-alpha). The primary role of TNF-alpha is inthe regulation of immune cells. It is able to induce apoptotic celldeath, to induce inflammation, and to inhibit tumorigenesis and viralreplication. Dysregulation of TNF-alpha production has been implicatedin a variety of human diseases, including cancer. Thus, modulation ofthe activity of inflammatory mediators, such as TNF-alpha, may havebeneficial implications in regulating the inflammatory-mediatedcarcinogenesis. Alternatively, modulation of TNF-alpha converting enzyme(TACE), which releases active TNF-alpha, may also have beneficialimplications in regulating inflammatory-mediated carcinogenesis. It iscontemplated that the antimicrobial agent of the present applicationwould be effective in individuals with or at risk of developing cancer.It is further contemplated that cancers at various carcinogenic stages,either in the precancerous stage, during the development of cancers orin the metastatic stages are included within the present application.

p53 (also known as protein 53 or tumor protein 53) is another well-knownmediator that is a tumor suppressor/pro-apoptotic protein important inmulticellular organisms. It regulates the cell cycle and, thus,functions as a tumor suppressor that is involved in preventing cancer.p53 is also a general inhibitor of inflammation that acts as anantagonist of nuclear factor kappaB (NFkappaB). Studies have shown thatp53, acting through suppression of NFkappaB, plays the role of a general“buffer” of innate immune response in vivo that is well consistent withits tumor suppressor function. This provides further evidence thatimmunomodulation may be an effective approach to mitigate or treatinflammatory-mediated carcinogenesis. It is contemplated that theantimicrobial agent of the present application would be effective inindividuals with or at risk of developing cancer. It is furthercontemplated that cancers at various carcinogenic stages, either in theprecancerous stage, during the development of cancers or in themetastatic stages are included within the present application.

The pro-apoptotic effects of p53 have also been associated with humanpapillomaviruses (HPVs) and the development of cancer. For example, theHPV type 16 oncoprotein, E6, complexes with and promotes degradation ofp53. Interestingly, HPV type 16 also appears to play a role in thedevelopment of certain malignancies. It is contemplated that apoptoticmediators may be involved in the regulation of cancer and thatimmunomodulation may be an effective approach.

The role of epidermal growth factor (EGF) and vascular endothelialgrowth factor (VEGF) in tumorigenesis has also been well documented. EGFis involved in the regulation of cell growth, proliferation anddifferentiation. Upregulation of EGF/EGFR (epidermal growth factorreceptor) activity leads to uncontrolled cell division, a predispositionto the development of cancer. VEGF is produced by cells that stimulatethe growth of new blood vessels, a process known as angiogenesis.Angiogenesis is necessary for the growth and metastasis of tumors andinhibition of VEGF impairs angiogenesis and disrupts metastatic spread.It is contemplated that immunomodulation may be an effective approach toregulate the development and/or progression of cancer initiated ormediated by the EGF and/or VEGF pathways. Interestingly, studies havedemonstrated that ethanol can induce structural and functionalalterations of the EGFR molecule, resulting in decreased EGF receptorbinding, and thereby impairing its receptor kinase activity and itsphysiological function. Thus, it is contemplated that the ethanolalcohol component within the antimicrobial agent of the presentapplication may be effective in regulating the development and/orprogression of cancer via modulation of the activity of the EGFRmolecule.

The present application demonstrates that the antimicrobial agent of thepresent application was able to modify and significantly and rapidlyimprove the course of leukoplakia (a pre-malignant lesion) or cancer,either in the carcinoma-in-situ stage or in the invasive carcinomastage. The development of the leukoplakia was in an infected area of thesubject diagnosed with chronic periodontitis, an inflammatory conditioncharacterized by chronic inflammation of the periodontal tissues that iscaused by accumulation of profuse amounts of dental plaque. Chronicperiodontitis is initiated Gram-negative and Gram-positive tooth andgingival-associated microbial biofilms that elicit a host response,which results in bone and soft tissue destruction. This disease isassociated with a variable microbial pattern. In response to endotoxinderived from periodontal pathogens, several osteoclast-related mediatorstarget the destruction of alveolar bone and supporting connective tissuesuch as the periodontal ligament. Some major drivers of this aggressivetissue destruction include matrix metalloproteinases (MMPs), cathepsins,and other osteoclast-derived enzymes. Although sub-antimicrobial dosesof antibiotics have been used to alter host response to the periodontalpathogens, it has been demonstrated that topical treatment usingdoxycycline or minocycline antibiotics leads to resistance of not onlyoral flora, but may colonize the patient in other body sites forpotential infection. Chlorhexidine oral application selects out moreresistant bacteria, e.g., methicillin resistant Staphylococcus aureus(MRSA), which could lead to persistent inflammation and transmission ofresistant pathogens. The present application demonstrates that theantimicrobial agent was effective at inhibiting and treating theleukoplakia condition and forms of squamous cell carcinoma, which arelikely secondary manifestations induced by the cytokines, chemokines andother inflammatory molecules present during an inflammatory reaction.

The role of viruses (and viral infections) in the pathogenesis ofcancers is another important medical research area. Human papillomavirus (HPV) is a member of the papillomavirus family of viruses that iscapable of infecting humans. HPV infections occur in the stratifiedepithelium of the skin or mucous membranes (such as in the cervix,vulva, vagina, penis, anus and oropharyngx). Persistent infection with“high-risk” HPV types may progress to precancerous lesions and invasivecancer. A growing number of studies have shown a link between HPVinfection and certain types of cancers (such as penile and analcancers). Further studies have also shown a link between a wide range ofHPV types and squamous cell carcinoma of the skin. It is contemplatedthat effective treatment of the leukoplakia condition (a form ofsquamous cell carcinoma) using the antimicrobial agent of the presentapplication may be mediated, in part, by its effect on any possibleunderlying viral infection or activity. The effect may also be mediatedby any possible underlying inflammatory activity.

Interestingly, the E6 and E7 proteins of HPV have been associated withpromotion of dysplasia and squamous cell carcinoma. In particular, theE6 protein is involved in numerous activities including inactivatingp53, blocking apoptosis, activating telomerase, disrupting celladhesion, polarity and epithelial differentiation, alteringtranscription and reducing immune recognition. The E6 protein containsfour cysteine arrays that constitute two relatively large zinc fingers,both of which are required for full function. It is contemplated thatthe antimicrobial agent of the present application may disrupt the zincfinger formation and stability of the E6 protein through its chelatingfunction in sequestering and binding of free Zn+2 ions. This may providean effective means for inhibiting or treating HPV-mediated carcinomas.

Another example of the role of viruses in the pathogenesis of cancers isdemonstrated by the Epstein-Barr virus (EBV), also called human herpesvirus 4 (HHV-4). It is known to be a cancer-causing virus of the herpesfamily, and is one of the most common viruses in humans. There is strongevidence that the virus has a primary role in the pathogenesis ofmultiple cancers, particularly Hodgkin's disease, Burkitt's lymphoma,nasopharyngeal carcinoma, and central nervous system lymphomasassociated with HIV. In cases of nasopharyngeal carcinoma, it iscontemplated that the antimicrobial agent of the present applicationwould be an effective means for inhibiting or treating the carcinoma viaits effect on any possible underlying viral infection or activity. Theeffect may also be mediated by any possible underlying inflammatoryactivity.

The antimicrobial agent of the present application may further provide ameans of significantly reducing the severity and shortening the courseof “cold sores” likely resulting from an outbreak of Herpes simplex.Accordingly, the antimicrobial agent of the present application may alsobe provided to inhibit or reduce a viral condition or disease. Theantimicrobial agent of the present application may also be provided totreat a viral condition or disease.

Another contemplated application of the antimicrobial agent of thepresent invention may be for the inhibition and/or treatment of cankersores (aphthous), a type of oral ulcer, which presents as a painful opensore inside the mouth or upper throat and is characterized by a break inthe mucous membrane. Once thought to be a herpes virus infection, theentire class of canker sores is now thought to be an aggregate of avariety of disease processes, each with the ability, in its own way, toproduce rapid but self-limiting destruction of mucous membranes,predominantly through immunologic and ischemic mechanisms. In someindividuals the ulcers are a secondary or hypersensitivity response toantigenic stimulus, especially foods), while in others they are aprimary autoimmune disorder. It is contemplated that the antimicrobialagent of the present application may be useful for the inhibition and/ortreatment of canker sores. The effect may be mediated by modulation ofany underlying viral infection or activity or inflammatory activity.

It is contemplated that the antimicrobial agent of the presentapplication may be used as adjunctive therapy in combination withexisting therapies. The term “adjunctive” is used interchangeably with“in combination” or “combinatorial” and are used where two or moretherapeutic or prophylactic regimens affect the treatment or preventionof the same disease. For example, the antimicrobial agent of the presentapplication may be used as adjunctive therapy for the management ofcancer. The antimicrobial agent of the present application may providesynergistic effects, both in anti-cancer efficacy and in control orreduction of side effects, such as toxicity from chemotherapy orradiation therapy and chemoresistance. The antimicrobial agent of thepresent application may provide a means of adjusting (e.g., reducing)the dosages from the existing therapies such that the desired effect isobtained without meeting the threshold dosage required to achievesignificant side effects. For example, the antimicrobial agent of thepresent application may be used as an adjunctive therapy to radiationtherapy which creates hydroxyl radicals and DNA damage to cancer cells,by potentially reducing the dose and/or duration of radiotherapy andincreasing efficacy with reduced toxicity. It is contemplated that suchadjunctive treatment may be achieved by way of simultaneous, sequentialor separate dosing from the existing therapies.

Accordingly, the antimicrobial agent of the present application may alsobe provided to inhibit or reduce a condition or disease resulting fromor associated with microbial growth or infection. The antimicrobialagent of the present application may also be provided to treat acondition or disease resulting from or associated with microbial growthor infection. Such conditions may include but is not limited toinflammatory diseases, viral diseases and cancer or pre-cancerousconditions.

The antimicrobial agent of the present application may provide a meansfor altering the chemical environment of target areas of human andanimal patients, especially in wounds, burns, surgical sites, andcatheter insertion sites, to prevent cell damage and/or toxicity by thepresence of a range of materials that are commonly found at such sites.Such materials often have a balance of beneficial and deleteriouseffects, depending on their concentrations and other factors. They areusually present in very low levels, generally in the micromolar or evenat the nanomolar levels (10⁻⁹) levels, and in some cases picomolar(10⁻¹²) levels. Accordingly, this application provides a series ofreactive possibilities at such levels because the combination ofcomponents in the antimicrobial agent are present at significantlyhigher levels (i.e., millimolar or molar), which has the effect ofdriving reactions, which would otherwise seem to be unfavorable, furthertowards completion.

It is contemplated that the antimicrobial agent of the presentapplication may be provided to target sites by means of a medicaldevice. Medical devices may be treated or coated with the antimicrobialagent of the present application and incorporated into medical anddental instruments including disposable or permanent or indwellingcatheters (e.g., central venous catheters, dialysis catheters, long-termtunneled central venous catheters, short-term central venous catheters,peripherally inserted central catheters, peripheral venous catheters,pulmonary artery catheters, urinary catheters, and peritonealcatheters), urinary devices, vascular grafts, vascular catheter ports,wound drain tubes, ventricular catheters, hydrocephalus shunts, heartvalves, heart assist devices (e.g., left ventricular assist devices),pacemaker capsules, incontinence devices, penile implants, vulvardevices, small or temporary joint replacements, urinary dilator,cannulas, elastomers, hydrogels, surgical instruments, dentalinstruments such as dental trays, tubings, such as intravenous tubes,breathing tubes, adhesives (e.g., hydrogel adhesives, hot-meltadhesives, silicone-based adhesives or solvent-based adhesives),bandages, orthopedic implants, and any other device used in the medicaland dental field. Medical devices also include any device which may beinserted or implanted into a human being or other animal, or placed atthe insertion or implantation site such as the skin near the insertionor implantation site. Medical devices further include surfaces ofequipment in operating rooms, emergency rooms, hospital rooms, clinics,and bathrooms.

It is also contemplated that the antimicrobial agent of the presentapplication may be effective as a spermicidal and antimicrobial agent,which could help prevent the spread of sexually transmitted diseases.Prevention of HPV transmission, along with vaccines, may markedly reducecervical cancer, as well as some vulvar and oral pharyngeal carcinomas.The antimicrobial agent of the present application may be administeredalone or in combination with one or more barrier methods ofcontraception, such as a diaphragm, sponge or condom.

The antimicrobial agent of the present application may be utilized as acleaner/antiseptic when applied to hands to reduce or prevent thetransmission of bacterial, fungal, viral and parasitic diseases,especially in a clinical environment.

The antimicrobial agent of the present application may provide a meansby which biofilms as formed by bacterial and fungal organisms aredisrupted and the embedded organisms in the biofilm, whether they arebiofilm formers or present by accidental inclusion, entrapment orotherwise, are killed or rendered non-viable and/or otherwisenon-threatening.

The antimicrobial agent of the present application may provide a meansby which bacterial and yeast spores are either killed outright in thespore stage, or are rendered ineffective by being unable to germinate,or by germination followed by rapid killing before their pathogenicpotential is expressed.

The antimicrobial agent of the present application may provide a meansby which quorum sensing (QS) mechanisms used by biofilm formingmicroorganisms is interrupted. While these mechanisms vary from organismto organism, and gram-positive organisms use a somewhat different QSsystem from gram-negative, in all cases there is a molecule or series ofmolecules which provide the QS function. In many cases, these areprotein molecules that are potentially susceptible to structural changesby reaction with the antimicrobial agent of the present application.Reactions such as hydrolysis, alcoholysis, esterification,transesterification, oxidation, protein denaturation, or chelation ofboth free ions and partially bound cations are likely possibilities.Substances which are targets for hydrolytic or alcoholytic destructionor disruption are, for example, acyl homoserine lactones (AHL's, the QSmolecules of gram-positive biofilm formers), and other lactone or estercomponents of gram-positive bacteria. Biofilms area are also known to bedisrupted by chelators, which it is believed, results from the bindingeffect of the chelator on divalent, trivalent or other cations necessaryfor the formation of the biofilm. Inclusion of one or more chelators,generally preferred but not limited to EDTA, provides this function tothe antimicrobial agent of the present application.

Candida Albicans

As biofilm studies proliferate and as their importance in human andanimal pathogenesis becomes more widely recognized, other points ofvulnerability become known. Often, these studies are organism-specificand thus their potential for generalized medical applications is notknown as yet. On the other hand, some involve such important pathogensthat even a narrow range of applicable species provides a window intomedically important treatments. For example, Mukherjee et al. have shownthat the enzyme alcohol dehydrogenase (Adh1p) restricts the ability ofCandida albicans to form biofilms on catheter surfaces via analcohol-based mechanism. Interestingly, although the Adh1p enzyme isnecessary for the formation of the biofilm, once the biofilm is producedby the planktonic C. albicans cells, the production of Adh1p issignificantly reduced relative to the planktonic quantity. Alsointerestingly, they found a significant change in the chemical activityof the Adh1p's conversion of acetaldehyde into ethanol when comparisonwas made between planktonic and biofilm C. albicans. In the planktonicform the enzyme is producing larger quantities of ethanol whereas in thebiofilm acetaldehyde quantities rise significantly. This finding seemsto imply that the biofilm-bound Adh1p is unable to process theacetaldehyde to ethanol, but does not explain the mechanism of thephenomenon. We can speculate, however, that by limiting the amount ofethanol in the biofilm, the C. albicans are down-regulating the Adh1pactivity which in turn fosters additional biofilm growth. Theantimicrobial agent of the present application is designed with a highlevel of ethanol, which alone provides significant activity against C.albicans through a Mukherjee-effect reduction of biofilm formation. Inaddition, our combinations of ingredients have already been shown to bequickly active against planktonic C. albicans (unpublished data). Thiscombination of high-level killing of planktonic cells and the ability toassist in prevention of biofilm formation by the few remaining viablecells would be expected to provide a patient with very substantialprotection against Candida infection of catheters and other implanteddevices.

This hypothesis is supported by the work of Baillie and Douglas. Theirwork developed physical conditions for growing Candida biofilms ofmaximal thickness and density and a study of the chemical composition ofthem. Under static conditions, the formation of a biofilm matrix isminimal but is greatly enhanced by the presence of a liquid flow. It isof interest that they found that the extent of the matrix formation didnot affect the susceptibility of the biofilms to different antifungaldrugs, including flucytosine and three azole compounds, even at levelsrepresenting many multiples of the planktonic MIC.

The independence of susceptibility by differing thicknesses/densities ofthe biofilm matrix is verified by Andes et al. where in Candida, theyalso cite data showing the importance of controlling or eliminatinginfections. Most of the candidemia cases involve catheters, and in thelargest reported study, 71% these cases implicated a catheter. Incatheter-related Candida bloodstream infections, 41% mortality was seenin patients whose catheter was retained. Andes et al. also stress thatthey observed a significant difference in the biofilm forming behaviorof Candida depending on the surface material of the catheter or otherplastic substrate. Even similar polymeric materials, e.g.,polyvinylchloride (PVC) exhibited differences when provided by differentmanufacturers.

This work is of particular interest to the Assignee in that the originalapplication all involves medical devices wherein the flow of liquid iseither minimal or non-existent. In those cases, where the biofilm matrixis not stimulated by flow, the tendency will be for the microorganismsto remain planktonic and therefore more vulnerable to the solution.

Staphylococcus Aureus

Another species receiving much medical attention recently and whichpotentially provides an alternative window into biofilm prevention ormitigation is Staphylococcus aureus. In particular MRSA, i.e.,methicillin resistant S. aureus, is particularly troublesome anddangerous. Caiazza and O'Toole have shown that the cell-to-cellinteraction promoter alpha-toxin, also called alpha-hemolysin, isrequired for biofilm formation. Other substances such as autolysin,teichoic acids and surface proteins are integral to the early formationof biofilm colonization, but alpha-toxin is essential. It appears thatthe alpha-toxin is required for the synthesis of polysaccharideintracellular adhesion (PIA). Alpha-toxin has been known for some time(see, for example, Bhakdi S, Tranum-Jensen J) and has been characterizedas a hydrophilic protein, MW=34 kD, and which is shown to be apore-former for the surface membranes of the organism.

In the application of the antimicrobial agent of the present applicationto mitigation of S. aureus biofilms, we would primarily expect thathydrogen peroxide would attack the alpha-toxin structure by oxidativebreaking of S—S bonds, oxidation of methionine methylthioether tosulfoxide, oxidation of free amino-groups and free hydroxide groups andthus disrupt the folding of the protein. Such changes would likely alsodisrupt the ability of the alpha-toxin to encourage biofilm formation.Other gram-positive bacteria, such as Streptococcus species, with somesimilar cell membrane structures, toxins and biofilms could be expectedto be susceptible to the action of the antimicrobial agent of thepresent application.

Gram-Negative Bacteria, Including Escherichia Coli, Klebsiella andPseudomonas Aeruginosa

Lipopolysaccharide, often called Lipid A, is an endotoxin known to be acontrolling factor necessary to the biogenesis of membrane lipids. LipidA is one of the highly toxic components released on the death of somebacterial cells and causes toxic shock in some cases. The structure ofLipid A is known (see, for example, Jia et al.). While the general corestructure is essentially invariant, a number of species-dependentmodifications are known. One highly hydrophobic section is formed from aseries (usually four chains) of fatty acyl esters or amides of theeither sugar-ring—bound OH's or sugar-ring bound NH's. These four chainshave hydroxyl groups in the 3′ positions, which are often also acylated,generating a large hydrophobic zone in the molecule, usually with twoadditional chains. Jia et al. report that one of the hydroxy groupsparticipates in the lipid trafficking across the outer bacterialmembrane (OM) by forming a palmitoyl ester at the 3′ hydroxide. Theformation of the palmitoyl ester provides a protective function,preventing host immune system attack, and controlling endotoxinformation.

This approach should be able to avoid the rapid generation of resistanceby the bacteria since it does not provide selective pressure forresistance development. Stopping or interfering with a fundamentalprocess leading to biofilm formation, could prevent a simple mutation orsimple series of mutations to bypass or counteract the genesis ofbiofilm. It is also important to note that the hypothesized processtakes place in the presence of the antimicrobial agent of the presentapplication which has already been shown to be toxicidal to the broadspectrum of tested microorganisms, including biofilm formers,non-biofilm formers and spores. For example against Bacillus cereusspores, the antimicrobial agent of the present application quicklycaused a rapid sporicidal reduction of greater than 6-log colony formingunits (CFU) (unpublished results). Because of the multitude of differentphysicochemical reactions triggered by the antimicrobial agent of thepresent application, survival of vegetative organisms or spores, even inprotective biofilms, would be extremely unlikely or more particularly,to reproduce. Additionally, the probability for development of geneticresistance would be even lower. Since the therapy of the antimicrobialagent of the present application may include topical application anddoes include antimicrobials agents used in systemic therapy, theantimicrobial agent of the present application would not promoteresistance to commonly used systemic antimicrobials.

Other effects relating to biofilms and their formation are candidatesfor the antimicrobial agent of the present application. For example, asmentioned above, biofilms, once formed, are able to reduce theeffectiveness of many known antibiotics by several orders of magnitude,compared to the therapeutic levels for planktonic forms of the samespecies. This problem is exacerbated in the case of some bacteria by thefact that they change their phenotypic presentation during and after theformation of the biofilm. For example, Pseudomonas aeruginosa displaysmultiple phenotypes during biofilm development, in fact at least fourstages were identified including one stage which involves thedevelopment and use of flagella not present in other phenotypes. Thus,in addition to the potential resistance to “traditional” antibioticsbecause of the presence of the physiochemical effects of the biofilmitself, it is clear that a range of endogenous changes in geneexpression are occurring, thus complicating the use of traditionalantibiotics or the search for new ones. The effect of changing theexpression of portions of the genome can also change the degree ofdiversity within a given species of biofilm, even though no externalstress or mutagen is applied. Such “variants”, as shown by Boles andSingh are generated by biofilms of Pseudomonas aeruginosa, Pseudomonasfluorescence, Vibrio cholera, Staphylococcus pneumonia andStaphylococcus aureus. They appear to be produced from wild-type (WT)organisms which are subject to endogenous oxidative stress. Addition ofanti-oxidants, e.g., N-acetyl cysteine or L-proline reduced oreliminated the ability of WT to produce the variants. And, to reiterate,fewer variants result in easier-to-control biofilms. Thus it might benecessary to “fine-tune” the antimicrobial agent of the presentapplication in some cases, particularly where the target organisms mightbe responding to the original treatment of the antimicrobial agent ofthe present application by generating variants. Cantin and Woods showedthat hydrogen peroxide can act in vitro with chloride ion (Cl-1) togenerate hypochlorous acid (HOCl), which in turn can react to formchloramines species in the presence of aminoglycosides like tobramycinor gentamicin. Using 5-thio-2-nitrobenzene as a model compound forsulfur-based oxidative stress or oxidative damage, they found thatdimethyl sulfoxide (DMSO) added to a solution containing HOCl protectedthe 5-thio-2-nitrobenzene from oxidation, but DMSO was unable to providethe same protective action in the presence of gentamicin or tobramycin.Since DMSO is known to have minimal toxic and other disruptive effectsto humans in low concentrations, DMSO and similar compounds representpotential additives to the antimicrobial agent of the presentapplication to fine-tune its oxidative properties. Similarly, it hasalso been shown that the oxidant products are hydrophilic. In the caseof the antimicrobial agent of the present application, then, the ethanolfraction of the mixture would become relatively more hydrophobic becauseof the migration of hydrophilic materials (i.e., ions and the like),towards the aqueous phase. The ethanol thus “freed” would become morepenetrating and more toxic to the lipid fractions, including the cellmembranes or cell walls of the biofilm formers and their EPS “fortress”.Additional deleterious effects on bacterial quorum sensing, bacterialcytotoxicity and proliferation and biofilm formation would be expected.

In intravenous infusion therapy involving catheters, clotting avoidanceand maintenance of sterility on the inner surfaces of the catheter portand lumen is essential. For that reason, use of flush solutions and locksolutions has become the standard of practice. The antimicrobial agentof the present application, in one embodiment, is a useful catheter locksolution. In this embodiment, the ingredients list would exclude anythickener or gellant and would reduce the peroxide level toapproximately 0.5%-1.0%.

The present application is further illustrated by the following specificexamples. The examples are provided for illustration only and should notbe construed as limiting the scope of the application in any way.

EXAMPLES Example 1 Preparation of the Antimicrobial Agent

The antimicrobial agents of the present application were prepared bymixing a low molecular weight alcohol, such as ethanol, a peroxide orperoxide-generating agent, such as hydrogen peroxide, and a chelatingagent, such as EDTA, at various concentrations.

Briefly, the desired amount of dry disodium EDTA dehydrate was measuredand added to a 50 mL flask. Next, the desired amount of hydrogenperoxide (H₂O₂) was added to the EDTA along with the desired amount ofsterile water. The test solution was mechanically mixed until the EDTAhad completely dissolved, during which the pH was monitored. Once thetest solution appeared translucent, the test solution was adjusted to apH of ˜7.4 using the desired amount of 1.0 M NaOH. Next, the desiredamount of ethanol was slowly added (dropwise) to the test solution untilcomplete dissolution. The resulting test solution was again adjusted toa pH of ˜7.4, if necessary, using the desired amount of 1.0 M NaOH.Sterile water was added, if necessary, until the total weight wasapproximately 40.00 g.

Table 1 illustrates the properties resulting from the differentcombinations in the antimicrobial agents of the present application.

TABLE 1 EtOH (% v/v) H₂O₂ (% v/v) EDTA (mg/mL) Properties 50 0 10Precipitation formed 50 0 15 Precipitation formed 50 0 25 Precipitationformed 52 0 15 Precipitation formed 54 0 10 Precipitation formed 58 0 10Precipitation formed 60 0 10 Precipitation formed 60 0 20 Precipitationformed 25 0.5 15 No precipitation 50 0.5 7.5 No precipitation 25 1 10 Noprecipitation 50 1 10 No precipitation 56 1 10 No precipitation 60 1 20No precipitation 50 1.5 5 No precipitation 50 1.5 10 No precipitation 501.5 15 No precipitation 35 2 20 No precipitation 58 2 10 Noprecipitation 60 2 20 No precipitation 50 3 10 No precipitation 54 3 10No precipitation 60 3 10 No precipitation 60 3 20 No precipitation 35 420 No precipitation 50 4 10 No precipitation 56 4 10 No precipitation 604 20 No precipitation 35 5 20 No precipitation 50 5 7.5 No precipitation50 5 9 No precipitation 50 5 10 No precipitation 54 5 10 Noprecipitation 56 5 10 No precipitation 20 6 40 No precipitation 40 6 40No precipitation 50 6 40 No precipitation 50 7 10 No precipitation 107.5 40 No precipitation 20 7.5 40 No precipitation 40 7.5 40 Noprecipitation

As illustrated in Table 1, in the absence of H₂O₂, the solubility ofEDTA in ethanol was very limited and resulted in precipitation of thesolution. However, when H₂O₂ was present, even at very lowconcentrations, stable solutions of EDTA in ethanol were readilyprepared with no precipitation being observed at either room temperatureor after storage at 0° C.

Optionally, a viscosity-increasing agent, such as hydroxyl propyl methylcellulose (HPMC), can be further added to the antimicrobial agents ofthe present application. Briefly, to prepare an antimicrobial agentcomprising HPMC, two premixes were initially prepared: Premix A andPremix B. In Premix A, which comprises 20.0 g of 3% USP H₂O₂ and 0.40 gdisodium EDTA dehydrate, the ingredients were combined and mechanicallymixed to complete dissolution. In Premix B, which comprises 0.27 g DowK15M HPMC and 5.00 g absolute ethanol, the ingredients were combined andmixed well to make a smooth slurry. The Premix A solution was placed ona vigorous mechanical stirrer (magnetic) and treated with the slurry ofPremix B from a disposable pipette. Care was taken to ensure that thepipette-filled slurry was representative of the whole, and to place thebolus from each pipette loading into the most active part of the vortex.The clear solution became cloudy immediately but did not clump or formvisible separation. Addition of Premix B took approximately 5 minutesand then the whole mixture was stirred for an additional hour. At theend of that time, the solution became cloudy or somewhat opaque, but onstanding for 15 minutes, the entrapped air had escaped and the solutionbecame clear. Stirring was reinitiated and 15.0 g of absolute ethanolwas slowly added to the mixture. The mixture was initially clear, but atapproximately 31% ethanol content (e.g., when approximately 8.3 g ofethanol had been added), the mixture became increasingly cloudy. Afterethanol addition was complete, the mixture was stirred for another 15minutes. The resulting thickened mixture had a pearlescent appearance,and could be used after mild shaking or simple stirring. The initialviscosity was approximately 330 cps (Brookfield LVF2 @ 30 rpm). Theviscosity leveled off at about 270-280 cps after 10 days of standing atroom temperature.

Example 2 Antimicrobial Activity of the Antimicrobial Agent

To illustrate the efficacy of the antimicrobial agents of the presentapplication, three representative pathogenic microorganisms were chosenfor the experiments. Candida albicans is a fungal species, methicillinresistant Staphylococcus aureus (MRSA) is a gram-positive bacterialspecies and Pseudomonas aeruginosa is a gram-negative bacterial species.

Each test microorganism was inoculated in Brain Heart Infusion broth(BHIB) and incubated at 37° C. for 20 h-36 h, yielding a culturecontaining minimally 10⁸ colony forming units per milliliter (CFU/mL).Determination of the initial CFU/mL of the culture was accomplishedusing dilution plating onto Brain-Heart Infusion agar (BHIA) followed byincubation at 37° C. for 24 h.

To test the efficacy of the antimicrobial agents of the presentapplication, 1 mL of culture was pipetted into 5 mL of antimicrobialagent solution yielding a 5:1 ratio. This combination of theantimicrobial agent solution and microorganism was immediately vortexedand allowed to sit without disruption for the desired time interval.Once the desired time interval had been completed, the antimicrobialagent solution underwent filtration.

A syringe filtration device with a removable membrane functioned tocatch any microorganisms, while the antimicrobial agent solution andbroth were allowed to pass through, ensuring that the microorganism werein contact with the antimicrobial agent solution for the desired amountof time. Using a 0.2 m syringe, 1 mL of the mixture of microorganismculture and antimicrobial agent solution was passed through the filter.Approximately 2 mL of sterile 0.85% saline was used to rinse anyresidual antimicrobial agent solution from the filter. The membrane wasthen sterilely removed and subsequently placed onto a BHIA plate andincubated for 24 h to allow for CFU determination. The membrane alloweddiffusion of the nutrients from the agar to any viable cells, resultingin formation of colonies. Any growth on the membrane was confirmed byremoval of a single colony, streaked for isolation and gram stained.Additionally, in the absence of growth, a swab was taken from themembrane and subcultured on BHIA.

Table 2 summarizes the efficacy of the different antimicrobial agentsolutions tested in killing C. albicans, MRSA and P. aeruginoa.

TABLE 2 EtOH H₂O₂ EDTA (% v/v) (% v/v) (mg/mL) C. albicans MRSA P.aeruginoa 25 0.5 15 Treatment time Treatment time Not tested 2 min -Lawn observed 2 min - Lawn observed 15 min - 64 CFU 15 min - Lawnobserved 1 hour - 0 CFU 1 hour - Lawn observed 4 hours - 0 CFU 4 hours -2 CFU 6 hours - 0 CFU 6 hours - N/A Initial culture Initial culture0.4-1 × 10⁸ CFU/mL 7.8-10.3 × 10⁷ CFU/mL 25 1 10 Treatment timeTreatment time Not tested 2 min - Lawn observed 2 min - Lawn observed 15min - 4 CFU 15 min - Lawn observed 1 hour - 0 CFU 1 hour - Lawn observed4 hours - 0 CFU 4 hours - 0 CFU 6 hours - 0 CFU 6 hours - 0 CFU Initialculture Initial culture 0.4-1 × 10⁸ CFU/mL 7.8-10.3 × 10⁷ CFU/mL 50 1 10Treatment time Treatment time Not tested 2 min - 0 CFU 2 min - 0 CFU 15min - 0 CFU 15 min - 0 CFU 1 hour - 0 CFU 1 hour - 0 CFU 4 hours - 0 CFU4 hours - 0 CFU 6 hours - 0 CFU 6 hours - 0 CFU Initial culture Initialculture 0.4-1 × 10⁸ CFU/mL 7.8-10.3 × 10⁷ CFU/mL 50 0.5 7.5 Treatmenttime Treatment time Treatment time 2 min - 0 CFU 2 min - 0 CFU 2 min - 0CFU 15 min - 0 CFU 15 min - 0 CFU 15 min - 0 CFU 1 hour - 0 CFU 1 hour -0 CFU 1 hour - 0 CFU 4 hours - 0 CFU 4 hours - 0 CFU 4 hours - 0 CFU 6hours - 0 CFU 6 hours - 0 CFU 6 hours - 0 CFU Initial culture Initialculture Initial culture 0.4-1 × 10⁸ CFU/mL 0.2-7 × 10⁸ CFU/mL 2.2-3.3 ×10⁹ CFU/mL No growth observed in No growth observed in subculturedfilter subcultured filter membranes membranes 50 6 40 Treatment timeTreatment time Treatment time 30 sec - 0 CFU 30 sec - 0 CFU 30 sec - 0CFU 60 sec - 0 CFU 60 sec - 0 CFU 60 sec - 0 CFU 2 min - 0 CFU 2 min - 0CFU 2 min - 0 CFU 15 min - 0 CFU 15 min - 0 CFU 15 min - 0 CFU 1 hour -0 CFU 1 hour - 0 CFU 1 hour - 0 CFU Initial culture Initial cultureInitial culture 1.8-2.2 × 10⁸ CFU/mL 4.8-7 × 10⁸ CFU/mL 2.8-3.3 × 10⁹CFU/mL No growth observed in No growth observed in subcultured filtersubcultured filter membranes membranes 60 3 10 Treatment time Treatmenttime Treatment time 30 sec - 0 CFU 30 sec - 1 CFU 30 sec - 0 CFU 60sec - 0 CFU 60 sec - 0 CFU 60 sec - 0 CFU 2 min - 0 CFU 2 min - 0 CFU 2min - 0 CFU 15 min - 0 CFU 15 min -0 CFU 15 min - 0 CFU 1 hour - 0 CFU 1hour - 0 CFU 1 hour - 0 CFU Initial culture Initial culture Initialculture 0.8-2.2 × 10⁷ CFU/mL 4.8-7 × 10⁸ CFU/mL 2.8-3.3 × 10⁹ CFU/mL Nogrowth observed in No growth observed in subcultured filter subculturedfilter membranes membranes

As illustrated in Table 2, antimicrobial agent solutions comprising lowconcentrations of ethanol were not as effective in killing C. albicansand MRSA (P. aeruginoa killing was not tested). However, in the presenceof higher concentrations of ethanol, there was a loss of viability ofall three strains tested, as demonstrated by the lack of growth at allexposure/treatment times, even as early as 30 seconds.

The combination of components within the antimicrobial agents of thepresent application demonstrated lethality and were effective in killingthree representative and deadly catheter-related blood stream infection(CRBSI) pathogens: Candida albican, MRSA, and Pseudomonas aeruginosa, inonly 30 seconds, even with very high bacterial inocula. Thus, theantimicrobial agents of the present application were superior to otherantimicrobial agents that offer only a limited spectrum of organisms,i.e., no bacterial or fungal spores, with treatment times of 10-15minutes, and frequently with lower inocula tested.

Example 3 Immunomodulatory Activity of the Antimicrobial Agent

To test whether the inflammatory milieu present during an infectionmight activate or be associated with cancer (and pre-cancerous)development and/or progression, a human subject that had developedchronic periodontitis and displayed leukoplakia lesions within the oralcavity was studied.

The human subject was diagnosed with chronic periodontitis, aninflammatory condition characterized by chronic inflammation of theperiodontal tissues that is caused by accumulation of profuse amounts ofdental plaque. Chronic periodontitis may be initiated by Gram-negativeand Gram-positive tooth and gingival-associated bacteria, usuallyanaerobic or microaerophilic organisms and biofilms that elicit a hostresponse, which results in bone and soft tissue destruction. Thisdisease is associated with a variable microbial pattern. In response toendotoxin derived from periodontal pathogens, several osteoclast-relatedmediators target the destruction of alveolar bone and supportingconnective tissue such as the periodontal ligament. Some major driversof this aggressive tissue destruction include matrix metalloproteinases(MMPs), cathepsins, and other osteoclast-derived enzymes.

In addition to the being diagnosed with chronic periodontitis, thesubject developed leukoplakia lesions within the oral cavity displayinginvasive squamous cell carcinoma with surrounding carcinoma-in-situ anddysplasia in the right mandibular gingival area. Visual inspection ofthe lesions appeared as white, translucent patches. Histologicalexamination of biopsy specimens from the affected areas revealedevidence of surface epithelium exhibiting extensive atypia withunderlying fibrous connective tissue. The epithelial cells showedevidence of loss of maturation, nuclear hyperchromatism and nuclearcrowding. The underlying connective tissue showed infiltration bylymphocytes, plasma cells and neutrophils, characteristic of aninflammatory reaction. The epithelium also showed signs of verucoushyperparakeratosis and orthokeratosis, irregular acanthosis and basilarhyperplasia with mild epithelial hyperplasia, suggestive of a stage ofproliferative verrucous leukoplakia, a form of squamous cell carcinoma.Previously, the subject's dentists had attempted to treat the chronicperiodontitis and leukoplakia with a succession of oral antibiotics,antifungal fluconazole and prolonged courses of chlorhexidine oralrinses, in addition to vigorous dental hygiene. All of these therapeuticmeasures failed significantly to improve his condition or preventprogression to oral cancer.

A single topical application of the antimicrobial agent of the presentapplication in solution form was applied to the right lower mandibulargingival area while the right upper mandibular gingival area remaineduntreated. Visual inspection of the antimicrobial agent-treated areaproduced noticeable reduction in both the size and severity of thelesion within approximately 10 hours. Continued daily or twice-dailyapplications of the antimicrobial agent solution to the right lowermandibular gingival area over 12 days were effective to further reducethe lesion to approximately 1 mm. Subsequent biopsy of the lesion andexamination by histological examination revealed no evidence of invasivecarcinoma, with only slight dysplasia being present. No signs ofinflammatory cells were observed post treatment. In contrast, thepathology of the untreated right upper mandibular gingival areacontinued to display signs of invasive squamous cell carcinoma and wasonly finally removed by surgery post treatment.

These results suggest that the antimicrobial agent of the presentapplication was effective as an immunomodulator in treating conditionssuch as invasive squamous cell carcinomas that may have developed as aresult of the pre-existing inflammatory condition.

In addition to visual inspection of the treated area, the subject'supper and lower mandibular areas were examined pre- and post-treatmentfor the following physical characteristics: bleeding, suppuration,plaque, calculus, pocket depth and clinical attrition. Of the 32 teethfrom the subject, 192 sites were examined for the physicalcharacteristics as summarized in Table 3 below.

TABLE 3 % % Sites (Pre- Post- Teeth Examined treatment) treatment) %Change Total Teeth 32 192 — — — Bleeding 19/0  36/0  19 0 19%improvement Suppuration 0/0 0/0 0 0 No change Plaque 0/0 0/0 0 0 Nochange Calculus 0/0 0/0 0 0 No change 1-3 mm 25/13 114/50  59 26 33%pocket depth improvement 4-5 mm 13/12 30/41 16 21 5% pocket depthworsening 6+ mm 4/4 6/5 3 3 No change pocket depth 1-3 mm 10/6  19/20 1010 No change clinical attrition 4-5 mm 21/13 66/27 34 14 20% clinicalimprovement attrition 6+ mm 22/12 65/49 34 26 8% clinical improvementattrition

Based on the clinical measurements, treatment with the antimicrobialagent of the present application improved many physical characteristicsof the subject including bleeding (by 19%), 1-3 mm pocket depth (by33%), 4-5 mm clinical attrition (by 20%) and 6+mm clinical attrition (by8%). There were no changes to the subject's 6+mm pocket depth and 1-3 mmclinical attrition and about a 5% worsening of the subject's 4-5 mmpocket depth. These clinical results further suggest that theantimicrobial agent of the present application was effective inimproving the overall oral health of the subject, which may be due toits role as a potent immunomodulator.

In order to confirm that the effect of the antimicrobial agent solutionwas immunomodulatory and not anti-viral, biopsy samples from the samehuman subject were examined for the presence of human papilloma virus(HPV). A complete screen, testing for 37 different HPV species wasnegative, including HPV-16 and 18, the most common causes of genitalcancers. Specifically, using a PCR amplified protocol in conjunctionwith a Luminex bead assay detection system, the following HPV specieswere tested: HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52,53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81,82, 83, 84, IS39 and CP6108. No HPV species were detected in the biopsysamples although human DNA was detected.

These results provide further evidence that the cancer was unlikely tohave been caused by viral infection of the most common type, although itdoes not rule out other viral causes.

Example 4 Anti-Viral Activity of the Antimicrobial Agent

The anti-viral activity of the antimicrobial agent of the presentapplication was also studied in two human subjects that had developedcold sores.

Two human subjects had developed cold sores, which were consistent witha clinical diagnosis of recurrent herpes simplex virus (HSV) on thelips.

In the first subject, topical applications of the antimicrobial agent ofthe present application in solution form was applied to the cold soreaffected area when the outbreak was producing an unbroken, fluid-filled,blister-like vesicle. Over the course of two days, significant shrinkingand reduction of the vesicle to very small size was observed. Thevesicle produced a small area of cracked skin that also healed rapidlywith no visible lingering effects.

In the second subject, topical applications of the antimicrobial agentof the present application in solution form was applied to the cold soreaffected area only after the vesicle had ruptured. The treated subjectexhibited reduced severity and much more rapid healing than would betypical for cold sores in that subject.

The first subject had another recurring episode of a blister arisingfrom a potential herpes simplex breakout on the lips and upon topicalapplication of the antimicrobial agent of the present application insolution form, significant reduction and shrinking of the vesicle wasobserved. Continued topical application of the antimicrobial agent ofthe present application resulted in continued shrinking and reduction ofthe vesicle and rapid healing with no visible scabbing of the skin area.

Since herpes simplex is a virus, this data provides evidence that theantimicrobial agent of the present application exhibited anti-viralactivity.

All references, including publications, patent applications and patents,cited herein are hereby incorporated by reference to the same extent asif each reference was individually and specifically indicated to beincorporated by reference and was set forth in its entirety herein.

Any combination of the above-described elements in all possiblevariations thereof is encompassed by the application unless otherwiseindicated herein or otherwise clearly contradicted by context.

The terms “a” and “an” and “the” and similar referents as used in thecontext of describing the application are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the applicationand does not pose a limitation on the scope of the application unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theapplication unless as much is explicitly stated.

The description herein of any aspect or embodiment of the applicationusing terms such as “comprising,” “having,” “including” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the application that “consistsof,” “consists essentially of,” or “substantially comprises” thatparticular element or elements, unless otherwise stated or clearlycontradicted by context (e.g., a composition described herein ascomprising a particular element should be understood as also describinga composition consisting of that element, unless otherwise stated orclearly contradicted by context). That said, the terms “comprising,”“having,” “including” or “containing” in the claims should be construedaccording to the conventional “open” meaning of those terms in thepatent law to include those elements enumerated as well as otherelements. Likewise, the terms “consisting of,” “consists of,” “consistsessentially of,” or “substantially comprises” should be construedaccording to the “closed” or “partially closed” meanings ascribed tothose terms in the patent law.

This application includes all modifications and equivalents of thesubject matter recited in the aspects or embodiments presented herein tothe maximum extent permitted by applicable law.

We claim:
 1. An antimicrobial solution comprising: (a) water; (b) fromabout 20% to about 60% by volume of ethanol; (c) from about 0.5% toabout 7.5% by volume of hydrogen peroxide; and (d) from about 5 mg/mL toabout 40 mg/mL of ethylenediamine tetraacetic acid (EDTA), acids ofEDTA, salts of EDTA, or any combination thereof.
 2. The solution ofclaim 1, wherein the concentration of ethanol is about 50% by volume. 3.The solution of claim 1, wherein the ethylenediamine tetraacetic acid(EDTA), acids of EDTA, salts of EDTA, or any combination thereof ispresent at a concentration of about 10 mg/mL.
 4. The solution of claim1, wherein the hydrogen peroxide is present at a concentration of about1.5% by volume.
 5. The solution of claim 1, further comprising aviscosity-increasing agent.
 6. The solution of claim 5, wherein theviscosity-increasing agent comprises hydroxypropyl methylcellulose(HPMC).
 7. A method of inhibiting or reducing biofilm formation,comprising: identifying a site in need of biofilm formation inhibitionof reduction; and applying the antimicrobial solution of claim 1 to thesite, wherein the site is selected from the group consisting of a woundsite, a catheter site, a surgical site, an injection site, a catheter, acatheter lumen, a thermal burn site, a chemical burn site, a radiationburn site, a skin lesion, oral sites, bony sites, anal sites, vaginalsites, cervical sites, vulvar sites, penile sites, ulcerated skin sites,acne sites, actinic keratosis sites, inflamed sites, irritated sites,gastric sites, gastrointestinal sites, esophageal sites,esophagogastrointestinal sites, intestinal sites, cardiac sites,vascular sites, nasal sites, nasopharyngeal sites, and aural sites.
 8. Amethod of treating, inhibiting or reducing a condition or disease,comprising administering to a subject a therapeutically effective amountof the antimicrobial solution of claim 1, wherein the condition ordisease is a bacterial condition or disease, a viral condition ordisease, an inflammatory condition or disease, a pre-malignant conditionor disease, a cancerous condition or disease, or immune responsecondition or disease.
 9. The method of claim 8, wherein the solution isadministered by topical application, intravenous injection,intraperitoneal injection or implantation, intramuscular injection orimplantation, intralesional injection, subcutaneous injection orimplantation, intradermal injection, suppositories, pessaries, entericapplication, or nasal route.
 10. The method of claim 8, wherein thesolution is administered to a site selected from the group consisting ofa wound site, a catheter site, a surgical site, an injection site, acatheter, a catheter lumen, a thermal burn site, a chemical burn site, aradiation burn site, a skin lesion, oral sites, bony sites, anal sites,vaginal sites, cervical sites, vulvar sites, penile sites, ulceratedskin sites, acne sites, actinic keratosis sites, inflamed sites,irritated sites, gastric sites, gastrointestinal sites, esophagealsites, esophagogastrointestinal sites, intestinal sites, cardiac sites,vascular sites, nasal sites, nasopharyngeal sites, and aural sites.